This grant request is to continue studies of lung ischemia reperfusion injury. Ischemia reperfusion represents a potentially important mechanism for lung damage associated with pulmonary embolism, lung transplantation, and a variety of syndromes resulting in decreased pulmonary blood flow. During the preceding period of grant support, we have demonstrated that oxidation of lung tissue cellular components represents an initial manifestation of ischemia/reperfusion. A major finding was that significant oxidative lung injury occurred during lung ischemia and was unrelated to tissue anoxia. This renewal application will evaluate the mechanisms and pathways for oxidative injury. To explain the INITIATING factor that leads to oxyradical production in the absence of preceding anoxia, we have proposed a new mechanism based on sensing of the altered mechanical events associated with ischemia. The hypothesis is that decreased flow results in alterations of the endothelial cell membrane leading to increased production of oxyradicals and delocalization of Fe++/Fe+++ from tissue iron stores. The transducer for the "sensing" of ischemia may be the flow-activated K+ channels. Ischemia/reperfusion and the role of membrane perturbations will be studied with the isolated perfused rat lung and with bovine pulmonary artery endothelial cells in culture. Oxidation of tissue components is measured by thiobarbituric acid reactive substance (TBARS), conjugated dienes, and protein carbonyls. Tissue oxidation and release of Fe++, H2O2, and NO will be measured during ischemia, as a function of perfusate K+, and with activators and inhibitors of membrane K+ channels. As a second major goal, an in vivo rat model of lung ischemia reperfusion injury for comparison with the isolated lung model will be produced by reversible ligation of a major pulmonary artery. Our hypothesis is that tissue oxidative injury will be evident during the ischemic period, with amplification during reperfusion associated with influx of polymorphonuclear leukocytes. The relative contribution of the ischemic and reperfusion periods in the genesis of injury and the role of tissue oxygenation will be evaluated. The third major goal will evaluate the hypothesis that lung antioxidant enzymes are a target for protein oxidation during ischemic injury nd that protection is afforded by augmentation of antioxidant defenses. Antioxidant defenses will be augmented through use of mercaptoproprionylglycine (MPG), a sulfhydryl-based antioxidant and by specific targeting of antioxidant enzymes to pulmonary endothelium through coupling to a monoclonal antibody specific for lung endothelial angiotensin converting enzyme (ACE). The results of this program will provide insights into the mechanisms for tissue injury associated with lung ischemia/reperfusion and will indicate potential protective agents to prevent tissue damage.